(1) Field of the Invention
This invention relates to a method for amplifying DNA, and in particular to an improved method for amplifying DNA by Polymerase Chain Reaction (PCR).
(2) Description of the Related Art
The PCR is an In vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. PCR is described in U.S. Pat. Nos. 4,683,195 and 4,683,202 by K. B. Mullis et al.
The PCR is a highly sensitive test for the detection of DNA in blood, especially in peripheral blood leukocytes. Thus, PCR is a potentially powerful tool for use in the diagnosis of infectious diseases caused by microorganisms, hereditary diseases, cancers, etc. In addition, PCR is particularly suitable for determining parent/child relationships as peripheral blood is frequently the specimen of choice in such a determination.
One disadvantage to using PCR is that impurities such as pigmentary compounds, proteins, sugars and unidentified compounds inhibit the reaction. Therefore, separation of the cells from materials and the subsequent extraction of DNA from the cells is necessary prior to amplification by PCR. In order to overcome this inhibition, cellular lysis can be accomplished with enzymes, detergents or chaotropic agents and traditionally, the subsequent extraction of the nucleic acid from the cellular lysate has involved using phenol or phenol-chloroform mixture. More recent methods of purifying the DNA include the removal of impurities by using ion exchange resins, glass filter or beads or agents for protein flocculation.
One method (and reagent kit) for removing impurities is commercially marketed under the name of "COLLECTAGENE" (AMRAD Operations Pty Ltd., Victoria, Australia). In this method, the sample is prepared as follows:
1. Anticoagulant-treated whole blood of mammalian origin is mixed with Magnetic Particles in a tube, whereby the leukocyte cells and the Magnetic Particles are conjugated.
2. The conjugated cells and Magnetic Particles are washed with phosphate buffer.
3. The conjugated cells are lysed with proteinase whereby the proteins are degraded and the DNA in the leukocyte cells is released.
4. The tube is then heated to inactivate the residual proteinase resulting in a solution of free leukocyte DNA which is ready for PCR.
Meanwhile, RNA-PCR is utilized for detecting and/or sequencing target RNA and thus has potential for use as diagnostic tests and medical or environmental analyses. For example RNA-PCR may be useful for detecting HCV (hepatitis C virus) which causes hepatitis, and HIV (human immunodeficiency virus) which causes AIDS.
During RNA-PCR, a DNA which has a complementary base structure, (cDNA), is first synthesized using the target RNA as a template in the presence of reverse transcriptase. Then the cDNA is amplified by PCR. Under current technology, when treating the RNA obtained from living organisms (in particular from blood), purification of the RNA is necessary. Basically, the RNA can be purified using the same as the purification method for DNA.
In both DNA and RNA purifications it is difficult to sufficiently remove the impurities and thus the succeeding DNA amplification frequently ends in failure, especially when the sample contains a few copies of DNA. In addition, the purification methods are lengthy, complex, and costly.
Although, improved DNA purification methods such as the "COLLECTAGENE" method described above, are shorter and simpler to remove impurities, however, the possibility of contaminating DNA still remains. Because PCR methods are so sensitive that one DNA molecule in the specimen can be amplified, contamination may cause serious problems. Therefore, a simpler pretreatment which has less possibility of contamination is desirable.
In addition, a simpler method is desirable especially, for everyday use in the clinical field.
The present invention addresses these and other problems associated with PCR amplification procedures and is believed to represent a significant advance in the art.
The object of the present invention is to provide a PCR method which can achieve a specific amplification in the presence of impurities.
Another object of the present invention is to provide a PCR method suitable for everyday use in the clinical field.
Another object of the present invention is to provide a suitable kit for PCR, particularly a kit for use with blood samples.
Another object of the present invention is to provide diagnostic methods for detecting HIV (human immunodeficiency virus) which causes AIDS, HCV (hepatitis C virus) or HBV (hepatitis B virus) which cause hepatitis, a septicemia causative bacteria, cancer or hereditary diseases, using PCR or RNA-PCR.
More particularly, the object of this invention is to provide an improved process of PCR for DNA contained in leukocytes in peripheral blood.